The insect immune system consists of both cellular and humoral elements that innately recognize broad classes of foreign intruders. While great progress has been made over the last several years in identifying humoral factors like antimicrobial peptides and the signaling pathways that regulate their synthesis, much less is known about control of cellular defense responses. This is due in large part to the small size of many insects which makes collection of hemocytes and identification of hemocyte-produced effector molecules difficult. It is also often difficult to conduct manipulative experiments on hemocyte-mediated defense responses in vivo or to isolate defined populations of hemocytes for use in experiments in vitro. This is particularly true for vector arthropods like mosquitoes where little information exists on the origins, differentiation or functions of different hemocyte types. Mosquito hemocytes are classified primarily on the basis of morphology with only limited data relating these morphological characters to functional features. In the case of critically important vectors, like Anopheles gambiae, virtually nothing is known about hemocyte ontogeny in any life stage. Here, our goal is to use hemocyte-like cell lines (4a-3B cells), the fully sequenced An. gambiae genome, and our knowledge of hemocyte lineage markers from other insects to develop a robust suite of characters for identifying An. gambiae hemocytes. Given the exploratory nature of the application and limited background available on An. gambiae hemocytes, we think it appropriate to submit the application as an R21 proposal. Specific aims are: 1. Clone and characterize subpopulations of 4a-3B cells, 2. Characterize candidate generic and gene specific markers in An. gambiae hemocytes and 4a-3B cells, and 3. Conduct a proteomic analysis of the subcloned 4a-3B cells to identify new candidate markers. The results of this study will provide essential tools for understanding the role of different hemocytes in cellular immunity of An. gambiae and likely other species of mosquitoes.